The present invention relates to the identification of the minimal ankyrin binding domain (MAB) responsible for the interactions between a Na,K-ATPase and ankyrin. The present invention also relates to a method of determining the minimal ankyrin binding domain and its three dimensional structure in other integral membrane proteins that associate or bind to ankyrin through the use of deletion analysis and carrier mediated crystallization.
Another aspect of the present invention relates to a method of screening for compounds, including small peptides or peptide analogs, that inhibit, promote or modulate the interaction of a Na,K-ATPase and ankyrin. Lastly, the present invention relates to a method of inhibiting, promoting or modulating the interaction between a Na,K-ATPase and ankyrin in the presence of a compound, including small peptides or peptide analogs, that inhibits, enhances or modulates the interaction between a Na,K-ATPase and a
Tethering interactions between the cytoplasmic domains of integral membrane proteins and other macromolecules, mediated by ankyrin or proteins containing ankyrin-like repeat structures, play fundamental roles in diverse biological activities including growth and development (1, 2, 3, 4, 5), protein trafficking (6, 7, 8, 9), the establishment and maintenance of cellular polarity (10, 11, 12, 13), cell adhesion (14, 15), signal transduction (2, 16, 17, 18, 19, 20), and mRNA transcription (21, 22). Ankyrin, including its many isoforms (reviewed in 23, 24), is also the most ubiquitous adapter protein mediating linkage of membrane proteins with the spectrin-based skeleton, both at the plasma membrane (reviewed in (25, 26) as well as with internal membrane compartments including the Golgi apparatus (6, 7, 8). A characteristic feature of most ankyrins is the presence of a variable number of well conserved 33-residue repetitive units (ankyrin repeats) that individually or in combination bind specifically a large repertoire of transmembrane proteins (reviewed in 24). Since no data on the structure of a ankyrin binding domain in an integral membrane protein exists, there is little understanding of how such broad but specific binding capacity is achieved, or of how so many different membrane proteins can interact with a single ankyrin molecule (27, 28).
The xcex1-subunit of Na,K-ATPase interacts specifically with ankyrin (12, 29). This interaction is required for the stabilization of Na,K-ATPase at the basolateral plasma membrane of epithelial cells, and for the transport of NaK-ATPase from the endoplasmic reticulum to the Golgi apparatus (8). Distinct regions of both cytoplasmic domains II and III of xcex1-Na,K-ATPase bind ankyrin in vitro. These sequences appear to act independently, and those in cytoplasmic domain II (residues 140-290) account for most of xcex1-Na,K-ATPase""s affinity for ankyrin (29, 30).
The present inventors have for the first time discovered the threedimensional structure of the minimal ankyrin binding domain of a integral membrane protein that binds to ankyrin. The present invention is based on the inventors"" discovery that an ankyrin 33 residue repeating motif, an L-shaped structure with protruding xcex2-hairpin tips, mediates specific macromolecular interactions in cytoskeletal, membrane and regulatory proteins. The association between ankyrin and xcex1-Na,K-ATPase, a ubiquitous membrane protein critical to vectorial transport of ions and nutrients, is required to assemble and stabilize Na,K-ATPase at the membrane. xcex1-Na,K-ATPase binds both red cell (ANK1) and Madin Darby Canine Kidney (MDCK) cell ankyrin (ANK3) predominately by residues 142-166, located within its second putative cytoplasmic domain. Fusion peptides of glutathione-S-transferase incorporating these residues bind specifically to ankyrin. The three-dimensional structure (2.6 xc3x85) of this minimal ankyrin-binding motif fusion peptide reveals a 7 residue loop with one charged hydrophilic face capping a double xcex2-strand. Comparison of this structure with putative ankyrin binding sequences in p53, CD44, neurofascin/LI, and the IP3-receptor suggests that the versatility and specificity of ankyrin binding to its ligands is achieved by interactions involving the xcex2-hairpin tips of the ankyrin repeats with 5-7 residue loops presented at the surface of other such macromolecules.